摘要【目的】本课题拟通过改造吉马烯A合酶上的氨基酸位点，提高吉马烯A合酶（Germacrene A Synthase，GAS）的催化活性，获得高产β-榄香烯的工程菌株。【方法】实验室前期构建了含有CwGAS的DNA的pET-28a载体，提取质粒后利用定点突变方法将R327A和K332G分别导入质粒，得到重组质粒，琼脂糖凝胶方法验证；将重组质粒转化入Trans-5a感受态大肠杆菌，利用PCR电泳验证重组质粒是否成功转入；之后将转化成功的Trans-5a感受态大肠杆菌进行提取质粒，琼脂糖凝胶方法验证；之后质粒转入BL21感受态细胞，利用PCR电泳验证重组质粒是否成功转入；成功转入后，加入IPTG进行诱导纯化，利用SDS-PAGE检测；通过GC-MS测β-榄香烯含量。【结果】R327A和K332G成功突变；Trans-5α转化与BL21转化成功，PCR电泳图显示均有条带；SDS-PAGE检测R327A突变与突变前蛋白比较，R327A在62KDA处比突变前的浅，而K322G的突变蛋白没有发现目标蛋白条带。【结论】R327-K的突变导致吉马烯A合酶活性下降，而K332-G的突变导致吉马烯A合酶活性下降  ，为进一步通过改造GCS吉马烯A合酶提高β-榄香烯提供了实验数据。88260

毕业论文关键词：温郁金；β-榄香烯；定点突变；吉马烯A合酶

Abstract 【Purpose】: By transforming the amino acid sites of Germacrene A synthase, the catalytic activity of Germacrene A synthase was improved, and the high yield β-elemene strain was obtained。【Method】: The laboratory has a complete GAS DNA pET-28a carrier, use the fixed-point mutation method to transfer R327A and K332G respectively into plasmid after the plasmid was extracted, obtain the recombinant plasmid;The recombinant plasmid was transformed into the Trans-5α sensing Escherichia coli。Then extract plasmid from the Trans-5α sensing Escherichia colis;Then transfer plasmid to BL21 cells。After successfully transferring, add IPTG to induce purification and use SDS-PAGE for detection。The content of β-elemene was determined by GC-MS。【Results】:R327A and K332G successfully mutated; Trans-5α and BL21 successfully transformed, the PCR electrophoresis diagram showed strips; SDS-PAGE detection of R327A mutations at 62KDA was shallow compared to pre-mutant proteins, and no target protein bands were found at K322G's mutant protein。【Conclusions】:The mutation of R327A and K332G resulted in the decrease of the activity of Germacrene A synthase, which provided experimental data for further improving β-elemene production by transforming the Germacrene A synthase。

Keyword: Curcuma wenyujin; β-elemene; site-directed mutagenesis; Germacrene A Synthase

目    录

1  引言 5

1。1  温郁金及β-榄香烯简介 5

1。2  合成生物学 5

1。3  生物合成途径 6

1。4  定点突变 6

1。5  本研究目的及其意义 8

2  实验部分 9

2。1  实验材料源-于,优W尔Y论L文.网wwW.youeRw.com 原文+QQ75201,8766及试剂 9

2。1。1  菌株、载体和试剂 9

2。1。2  仪器设备 9

2。1。3  试剂及试剂盒 9

2。2  实验操作方法及步骤 11

2。2。1  提取质粒 11

2。2。2  定点突变